The present invention relates to the discovery of molecules that inhibit viral infection and promote an immune response to a pathogen. More specifically, the invention disclosed herein concerns molecules that interact with a FPR class receptor, inhibit HIV infection, and stimulate an inflammatory response in a subject.
Human monocytes express a wide variety of seven transmembrane (STM), G-protein coupled receptors including chemokine receptors and receptors for classic chemotactic factors such as the bacterial chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (fMLP), activated complement component 5 (C5a), and leukotriene B4 (LTB4). (Murphy. Annu Rev Immunol, 12:593 (1994); Murphy, xe2x80x9cThe N-formyl peptide chemotactic receptors,xe2x80x9d Chemoattractant ligands and their receptors, CRC Press, Boca Raton, 1996:269; and Prossnitz and Ye, Pharmacol Ther, 74:73 (1997)). Both classical chemoattractants and chemokines bind and activate these G-protein-coupled, STM receptors, which in turn, induce signaling cascades that promote cellular calcium (Ca2+) mobilization, phosphoinositide hydrolysis, chemotaxis, and activation of mitogen-activated protein kinase. (Schiffmann et al., Proc. Natl. Acad. Sci. U.S.A., 72:1059-1062 (1975); Oppenheim et al., Annu. Rev. Immunol., 9:617-648 (1991); Murphy, xe2x80x9cThe N-formyl peptide chemotactic receptors,xe2x80x9d Chemoattractant ligands and their receptors, CRC Press, Boca Raton, vol. 269 (1996); and Balkwill, J. Viral Hepatitis, 5:1-14 (1998)). Cells activated in this manner are essential participants in a host immune response to a pathogen.
The FPR class receptors are G-protein-coupled, STM receptors that bind the chemoattractant fMLP and are involved in monocyte chemotaxis and the induction of a host immune response to a pathogen. The prototype FPR class receptor, FPR, binds fMLP with high affinity and is activated by low concentrations of fMLP. The binding of FPR by fMLP induces a cascade of G protein-mediated signaling events leading to phagocytic cell adhesion, chemotaxis, release of oxygen intermediates, enhanced phagocytosis and bacterial killing, as well as MAP kinase activation and gene transcription. (Krump et al., J Biol Chem 272:937 (1997); Prossnitz et al., Pharmacol Ther 74:73 (1997); Murphy, Annu. Rev. Immuno. 12: 593 (1994); and Murphy, The N-formyl peptide chemotactic receptors, Chemoattractant ligands and their receptors. CRC Press, Boca Raton, p. 269 (1996)). Another FPR class receptor is the highly homologous variant of FPR, named FPRL1 (also referred to as FPRH2 and LXA4R). FPRL1 was originally cloned as an orphan receptor (Murphy et al., J. Biol. Chem., 267:7637-7643 (1992); Ye et al., Biochem. Biophys. Res. Commun., 184:582-589 (1992); Bao et al., Genomics, 13:437-440 (1992); Gao, J. L. and P. M. Murphy, J. Biol. Chem., 268:25395-25401 (1993); and Nomura et al., Int. Immunol., 5:1239-1249 (1993)) but was subsequently found to mediate Ca2+ mobilization in response to high concentrations of fMLP. (Ye et al., Biochem. Biophys. Res. Commun., 184:582-589 (1992); and Gao, J. L. and P. M. Murphy, J. Biol. Chem., 268:25395-25401 (1993)).
The chemokine receptor CCR5 is another G-protein-coupled, STM receptor and is a major fusion-cofactor exploited by most primary isolates of the human immunedeficiency virus type 1 (HIV-1). (Horuk, Immunol Today, 20:89 (1999); Dimitrov and Broder, xe2x80x9cHIV and Membrane Receptors,xe2x80x9d HIV and membrane fusion: Medical Intelligence Unit, Landes Bioscience, Austin, Tex., 1997:99; and Berger, AIDS 11, Suppl A:S3 (1997)). Individuals that fail to express CCR5 are largely resistant to HIV-1 infection. (Paxton, et al., Nat Med, 2:412 (1996); Dean, et al., Science, 273:1856 (1996)). Due to its prominent role in HIV-1 fusion and entry, investigators have foccussed considerable reasearch on developing molecules that interrupt the interaction between the HIV-1 envelope and CCR5. Chemokine ligands and antibodies specific for CCR5, for example, have been shown to inhibit HIV-1 entry and replication. (Cocchi et al., Science, 270:1811 (1995); Wu et al., J Exp Med, 186: 373 (1997); Proudfoot et al., J Biol Chem, 271:2599 (1996); Arenzana-Seisdedos et al., Nature, 383:400 (1996); Gong et al., J Biol Chem, 273:4289 (1998)).
One aspect of the invention concerns novel ligands for FPR class receptors that modulate a host immune response to a pathogen. Another aspect of the invention concerns the discovery that activated FPR class receptors promote phosphorylation and downregulation of CCR5, which, in turn, inhibits HIV infection. Several different ligands were found to interact with FPR class receptors and stimulate Ca+2 mobilization and chemotaxis in phagocytes. It was also found that the interaction of several of these ligands with the FPR class receptors resulted in the phosphorylation and downregulation of CCR5 and/or the inhibition of HIV infection.
In initial experiments, it was observed that binding of fMLP to an FPR class receptor resulted in phosphorylation and downregulation of CCR5 and the inhibition of HIV infection. Other experiments revealed that a synthetic peptide derived from the V3 region of gp120 (called xe2x80x9cV3 peptidexe2x80x9d), a synthetic peptide corresponding to a region in the C-terminus of the ectodomain of gp41 (called xe2x80x9cT20/DP178xe2x80x9d or xe2x80x9cT20xe2x80x9d), and serum amyloid A (xe2x80x9cSAAxe2x80x9d) activated FPR class receptors and induced the phosphorylation of CCR5. Additionally, it was found that a synthetic hexapeptide known as the leukocyte activating peptide or xe2x80x9cW peptidexe2x80x9d activated an FPR class receptor, induced Ca+2 mobilization and chemotaxis in phagocytes, and inhibited HIV-1 infection.
Embodiments of the invention include methods of identifying agents that inhibit HIV infection by interacting with an FPR class receptor. One approach involves techniques in rational drug design, whereby, compounds that resemble ligands for FPR class receptors (also referred to as xe2x80x9cbinding partnersxe2x80x9d) are designed and created using computer based homology searching, protein modeling, and combinatorial chemistry. These candidate binding partners are then evaluated for the ability to phosphorylate CCR5 and/or inhibit HIV infection in xe2x80x9cbinding partner characterization assaysxe2x80x9d. Agents that inhibit HIV infection are identified by their ability to phosphorylate CCR5 and/or inhibit HIV infection in the binding partner characterization assays. By another approach, random libraries of molecules are contacted to cells expressing CD4, CCR5, and an FPR class receptor and agents that inhibit HIV infection are identified by their ability to phosphorylate CCR5 and/or inhibit HIV infection. Still another approach utilizes two hybrid systems (e.g., yeast or mammalian two-hybrid systems) to identify agents that inhibit HIV infection. Many of these approaches are amenable to high throughput analysis.
Binding partners for FPR class receptors are preferably formulated into pharamceuticals and administered to subjects in need of a medicament that stimulates an immune response to a pathogen and/or treats and prevents HIV infection. Preferably, molelcules that interact with FPR class receptors are provided to human dendritic cells (DC), which express both FPR class receptors and CCR5. Further, since DCs present in human mucosal tissues are implicated as a major target cell for HIV, molecules of the invention can be delivered to the mucosal tissues (e.g., anal-vaginal mucosa) so as to reduce the risk of HIV transmission. These pharmaceuticals can be delivered by any conventional route including, but not limited to, transdermal, topical, parenteral, gastrointestinal, transbronchial, and transalveolar. A preferred application concerns the use of binding partners in a coating for medical devices.
Methods of inducing a host immune response to a pathogen and methods of inhibiting HIV infection are also embodiments. Accordingly, a method of inducing a host immune response to a pathogen involves identifying a subject in need of a compound that interacts with an FPR class receptor and providing said subject a therapeutically sufficient amount of V3 peptide or W peptide or a fragment, derivative, or modification thereof. A method of inhibiting HIV infection, according to one aspect of the invention, involves identifying a subject at risk for contracting HIV infection or a subject already afflicted with HIV infection and providing said subject a therapeutically sufficient amount of a binding partner for a FPR class receptor. Additional embodiments of the invention include a method of inducing phosphorylation of CCR5, a method inhibiting HIV fusion, and a method of treating and preventing HIV infection. These methods involve identifying a subject in need of a molecule that induces phosphorylation of CCR5 or a molecule that inhibits HIV fusion or a molecule that treats and prevents HIV infection, respectively, and administering to said subject a therapeutically sufficient amount of a binding partner for a FPR class receptor.